Use of SDF-1 to improve ischemic myocardial function

ABSTRACT

A method is provided for increasing trafficking of endothelial progenitor cells to an ischemic myocardium in a subject&#39;s heart comprising administering to the subject&#39;s heart an amount of Stromal-Derived Factor-1 (SDF-1).

This application is a continuation of U.S. Ser. No. 11/234,879, filedSept. 22, 2005, now U.S. Pat. No. 7,662,392, issued Feb. 16, 2010, whichis a continuation of U.S. Ser. No. 10/220,554, filed Mar. 4, 2003, nowabandoned, which is a continuation of PCT International ApplicationPCT/US01/18399, filed Jun. 5, 2001, which is a continuation-in-part andclaims benefit of U.S. Ser. No. 09/587,441, filed Jun. 5, 2000, nowabandoned, the contents of each of which are hereby incorporated byreference in their entireties into this application.

Throughout this application, various references are referred to withinparentheses. Disclosures of these publications in their entireties arehereby incorporated by reference into this application to more fullydescribe the state of the art to which this invention pertains. Fullbibliographic citation for these references may be found at the end ofthis application, preceding the claims.

BACKGROUND OF THE INVENTION

Left ventricular remodeling after myocardial infarction is a major causeof subsequent heart failure and death. The capillary network cannot keeppace with the greater demands of the hypertrophied but viablemyocardium, resulting in myocardial death and fibrous replacement. Thefirst series of experiments of the present invention, described below,show that human adult bone marrow contains endothelial cell precursorswith phenotypic and functional characteristics of embryonichemangioblasts, and that these can be mobilized, expanded, and used toinduce infarct bed vasculogenesis after experimental myocardialinfarction. The neo-angiogenesis results in significant and sustainedincrease in viable myocardial tissue, reduction in collagen deposition,and improved myocardial function. The use of cytokine-mobilizedautologous human bone marrow-derived angioblasts for revascularizationof myocardial infarct tissue, alone or in conjunction with currentlyused therapies, offers the potential to significantly reduce morbidityand mortality associated with left ventricular remodelingpost-myocardial infarction.

Although prompt reperfusion within a narrow time window hassignificantly reduced early mortality from acute myocardial infarction,post-infarction heart failure is increasing and reaching epidemicproportions (1). Left ventricular remodeling after myocardialinfarction, characterized by expansion of the initial infarct area,progressive thinning of the wall surrounding the infarct, and dilationof the left ventricular lumen, has been identified as a major prognosticfactor for subsequent heart failure (2, 3). This process is accompaniedby transcription of genes normally expressed only in the fetal state,rapid and progressive increase in collagen secretion by cardiacfibroblasts, deposition of fibrous tissue in the ventricular wall,increased wall stiffness, and both diastolic and systolic dysfunction(4, 5). Hypoxia directly stimulates collagen secretion by cardiacfibroblasts, while inhibiting DNA synthesis and cellular proliferation(6). In animal models, late reperfusion following experimentalmyocardial infarction at a point beyond myocardial salvage significantlybenefits remodeling (7). Moreover, the presence of a patent infarctrelated artery is consistently associated with survival benefits in thepost-infarction period in humans (8). This appears to be due to adequatereperfusion of the infarct vascular bed which modifies the ventricularremodeling process and prevents abnormal changes in wall motion (9).

Successful reperfusion of non-cardiac tissues rendered ischemic inexperimental animal models has recently been demonstrated by use ofeither circulating or bone marrow-derived cellular elements (10-13).Although the precise nature of these cells was not defined in thesestudies, the presence of precursor cells in both adult human circulationand bone marrow which have the capability to differentiate intofunctional endothelial cells, a process termed vasculogenesis (14-16),has been shown. In the pre-natal period, precursor cells derived fromthe ventral endothelium of the aorta in human and lower species havebeen shown to give rise to cellular elements involved in both theprocesses of vasculogenesis and hematopoiesis (17, 18). These cells havebeen termed embryonic hemangioblasts, are characterized by expression ofCD34, CD117 (stem cell factor receptor), Flk-1 (vascular endothelialcell growth factor receptor-2, VEGFR-2), and Tie-2 (angiopoietinreceptor), and have been shown to have high proliferative potential withblast colony formation in response to VEGF (19-22). The subsequentproliferation and differentiation of embryonic hemangioblasts toadult-type pluripotent stem cells appears to be related to co-expressionof the GATA-2 transcription factor, since GATA-2 knockout embryonic stemcells have a complete block in definitive hematopoiesis and seeding ofthe fetal liver and bone marrow (23). Moreover, the earliest precursorof both hematopoietic and endothelial cell lineage to have diverged fromembryonic ventral endothelium has been shown to express VEGF receptorsas well as GATA-2 and alpha4-integrins (24). The first series ofexperiments of the present invention shows that GATA-2 positive stemcell precursors are also present in adult human bone marrow, demonstrateproperties of hemangioblasts, and can be used to induce vasculogenesis,thus preventing remodeling and heart failure in experimental myocardialinfarction.

Growth of new vessels from pre-existing mature endothelium has beentermed angiogenesis, and can be regulated by many factors includingcertain CXC chemokines (47-50). In contrast, vasculogenesis is mediatedby bone marrow-derived endothelial precursors (51-53) with phenotypiccharacteristics of embryonic angioblasts and growth/differentiationproperties regulated by receptor tyrosine kinases such as vascularendothelial growth factor (VEGF) (54-57). Therapeutic vasculogenesis(58-61) has the potential to improve perfusion of ischemic tissues,however the receptor/ligand interactions involved in selectivetrafficking of endothelial precursors to sites of tissue ischemia arenot known. The second series of experiments of the present invention,described below, show that vasculogenesis can develop in infarctedmyocardium as a result of interactions between CXC receptors on humanbone marrow-derived angioblasts and ELR-positive CXC chemokines inducedby ischemia, including IL-8 and Gro-alpha. Moreover, redirectedtrafficking of angioblasts from the bone marrow to ischemic myocardiumcan be achieved by blocking CXCR4/SDF-1 interactions, resulting inincreased vasculogenesis, decreased myocardial death and fibrousreplacement, and improved cardiac function. The results of theexperiments indicate that CXC chemokines, including IL-8, Gro-alpha, andstromal-derived factor-1 (SDF-1), play a central role in regulatingvasculogenesis in the adult human, and suggest that manipulatinginteractions between CXC chemokines and their receptors on bonemarrow-derived angioblasts can lead to optimal therapeuticvasculogenesis and salvage of ischemic tissues. The third series ofexperiments, described below, show that CC chemokines also play a rolein mediating angioblast chemotaxis to ischemic myocardium.

The angiogenic response during wound repair or inflammation is thoughtto result from changes in adhesive interactions between endothelialcells in pre-existing vasculature and extracellular matrix which areregulated by locally-produced factors and which lead to endothelial cellmigration, proliferation, reorganization and microvessel formation (70).The human CXC chemokine family consists of small (<10 kD)heparin-binding polypeptides that bind to and have potent chemotacticactivity for endothelial cells. Three amino acid residues at theN-terminus (Glu-Leu-Arg, the ELR motif) determine binding of CXCchemokines such as IL-8 and Gro-alpha to CXC receptors 1 and 2 onendothelial cells (49,71), thus promoting endothelial chemotaxis andangiogenesis (47-48). In contrast, CXC chemokines lacking the ELR motifbind to different CXC receptors and inhibit growth-factor mediatedangiogenesis (49-72). Although SDF-1, an ELR-negative CXC chemokine, isa potent inducer of endothelial chemotaxis through interactions withCXCR4 (73), its angiogenic effects appear to be limited to thedeveloping gastrointestinal tract vascular system (50).

Vasculogenesis first occurs during the pre-natal period, withhaemangioblasts derived from the human ventral aorta giving rise to bothendothelial and haematopoietic cellular elements (74,75). Similarendothelial progenitor cells have recently been identified in adulthuman bone marrow (52-53), and shown to have the potential to inducevasculogenesis in ischemic tissues (59-62). However, the signals fromischemic sites required for chemoattraction of such bone marrow-derivedprecursors, and the receptors used by these cells for selectivetrafficking to these sites, are unknown. Following myocardial infarctiona process of neoangiogenesis occurs (62,63), but is insufficient tosustain viable tissue undergoing compensatory hypertrophy, leading tofurther cell death, expansion of the initial infarct area, and collagenreplacement (64-66). This process, termed remodeling, results inprogressive heart failure (67-69). In the experiments described below, anude rat model of myocardial infarction was used to investigate whetherCXC chemokines containing the ELR motif regulate migration of human bonemarrow-derived angioblasts to sites of tissue ischemia. Moreover, sinceselective bone marrow homing and engraftment of haematopoieticprogenitors depends on CXCR4 binding to SDF-1 expressed constitutivelyin the bone marrow (76-78), whether interruption of CXCR4/SDF-1interactions could redirect trafficking of human bone marrow-derivedangioblasts to sites of tissue ischemia, thereby augmenting therapeuticvasculogenesis, was examined. The results of the experiments indicatethat CXC chemokines, including IL-8, Gro-alpha, and SDF-1, play acentral role in regulating human adult bone marrow-dependentvasculogenesis. Further, the fourth series of experiments describedbelow show that stem cells can induce angiogenesis in peri-infarcttissue.

SUMMARY OF THE INVENTION

This invention provides a method of stimulating vasculogenesis inischemia-damaged tissue of a subject comprising:

-   -   (a) removing stem cells from a location within the subject;    -   (b) recovering endothelial progenitor cells from the stem cells        removed in step (a); and    -   (c) introducing the endothelial progenitor cells from step (b)        into a different location within the subject such that the        endothelial progenitor cells stimulate vasculogenesis in the        subject's ischemia-damaged tissue.

This invention also provides the instant method, wherein subsequent tostep (b), but before step (c), the endothelial progenitor cells areexpanded by contacting them with a growth factor.

This invention also provides the instant method, wherein the growthfactor is a cytokine.

This invention also provides the instant method, wherein the cytokine isVEGF, FGF, G-CSF, IGF, M-CSF, or GM-CSF.

This invention also provides the instant method, wherein the growthfactor is a chemokine.

This invention also provides the instant method, wherein the chemokineis Interleukin-8.

This invention also provides the instant method, wherein the endothelialprogenitor cells are separated from other stem cells before expansion.

This invention also provides the instant method, wherein theischemia-damaged tissue is myocardium.

This invention also provides the instant method, wherein theischemia-damaged tissue is nervous system tissue.

This invention also provides the instant method, wherein the stem cellsare removed from the subject's bone marrow.

This invention also provides the instant method, wherein the removal ofthe stem cells from the bone marrow is effected by aspiration from thesubject's bone marrow.

This invention also provides the instant method, wherein the removal ofthe stem cells from the subject is effected by a method comprising:

-   -   (a) introducing a growth factor into the subject to mobilize the        stem cells into the subject's blood; and    -   (b) removing a sample of blood containing the stem cells from        the subject.

This invention also provides the instant method, wherein the growthfactor is introduced into the subject subcutaneously, orally,intravenously or intramuscularly.

This invention also provides the instant method, wherein the growthfactor is a chemokine that induces mobilization.

This invention also provides the instant method, wherein the chemokineis Interleukin-8.

This invention also provides the instant method, wherein the growthfactor is a cytokine.

This invention also provides the instant method, wherein the cytokine isG-CSF, M-CSF, or GM-CSF.

This invention also provides the instant method, wherein the endothelialprogenitor cells are recovered based upon their expression of CD117.

This invention also provides the instant method, wherein the endothelialprogenitor cells are recovered based upon their expression of a GATA-2activated gene product.

This invention also provides the instant method, wherein the endothelialprogenitor cells are recovered based upon their expression of one ormore of CD34, VEGF-R, Tie-2, GATA-3 or AC133.

This invention also provides the instant method, wherein the subject hassuffered or is suffering from one or more of the following: myocardialinfarction, chronic heart failure, ischemic heart disease, coronaryartery disease, diabetic heart disease, hemorrhagic stroke, thromboticstroke, embolic stroke, limb ischemia, or another disease in whichtissue is rendered ischemic.

This invention also provides the instant method, wherein step (a) occursprior to the subject suffering ischemia-damaged tissue and wherein step(c) occurs after the subject has suffered ischemia-damaged tissue.

This invention also provides the instant method, wherein the endothelialprogenitor cells are frozen for a period of time between steps (b) and(c).

This invention also provides the instant method, wherein the endothelialprogenitor cells are frozen for a period of time after being expandedbut before step (c) is performed.

This invention also provides the instant method, wherein the endothelialprogenitor cells are introduced into the subject by injection directlyinto the peripheral circulation, heart muscle, left ventricle, rightventricle, coronary artery, cerebro-spinal fluid, neural tissue,ischemic tissue, or post-ischemic tissue.

This invention also provides the instant method, further comprisingadministering to the subject one or more of the following: an inhibitorof Plasminogen Activator Inhibitor, Angiotensin Converting EnzymeInhibitor or a beta blocker, wherein such administration occurs priorto, concomitant with, or following step (c).

This invention also provides a method of stimulating angiogenesis inperi-infarct tissue in a subject comprising:

-   -   (a) removing stem cells from a location within a subject;    -   (b) recovering endothelial progenitor cells from the stem cells        removed in step (a);    -   (c) expanding the endothelial progenitor cells recovered in        step (b) by contacting the progenitor cells with a growth        factor; and    -   (d) introducing the expanded endothelial progenitor cells from        step (c) into a different location in the subject such that the        endothelial progenitor cells stimulate angiogenesis in        peri-infarct tissue in the subject.

This invention also provides a method of selectively increasing thetrafficking of endothelial progenitor cells to ischemia-damaged tissuein a subject comprising:

-   -   (a) administering endothelial progenitor cells to a subject; and    -   (b) administering a chemokine to the subject so as to thereby        attract the endothelial progenitor cells to the ischemia-damaged        tissue.

This invention also provides the instant method, wherein the chemokineis administered to the subject prior to administering the endothelialprogenitor cells.

This invention also provides the instant method, wherein the chemokineis administered to the subject concurrently with the endothelialprogenitor cells.

This invention also provides the instant method, wherein the chemokineis administered to the subject after administering the endothelialprogenitor cells.

This invention also provides the instant method, wherein the chemokineis a CXC chemokine.

This invention also provides the instant method, wherein the CXCchemokine is selected from the group consisting of Interleukin-8,Gro-Alpha, or Stromal-Derived Factor-1.

This invention also provides the instant method, wherein the chemokineis a CC chemokine.

In an embodiment of this invention the CC chemokine is selected from thegroup consisting of RANTES, EOTAXIN, MCP-1, MCP-2, MCP-3, or MCP-4.

This invention also provides the instant method, wherein the chemokineis administered to the subject by injection into the subject'speripheral circulation, heart muscle, left ventricle, right ventricle,coronary arteries, cerebro-spinal fluid, neural tissue, ischemic tissue,or post-ischemic tissue.

This invention also provides a method of increasing trafficking ofendothelial progenitor cells to ischemia-damaged tissue in a subjectcomprising inhibiting any interaction between Stromal-Derived Factor-1and CXCR4.

This invention also provides the instant method, wherein interactionbetween Stromal-Derived Factor-1 (SDF-1) and CXCR4 is inhibited byadministration of an anti-SDF-1 or an anti-CXCR4 monoclonal antibody tothe subject.

This invention also provides the instant method, further comprisingadministering to the subject an angiotensin converting enzyme inhibitor,an AT₁-receptor blocker, or a beta blocker.

This invention also provides a method of reducing trafficking ofendothelial progenitor cells to bone marrow in a subject comprisinginhibiting production of Stromal-Derived Factor-1 in the subject's bonemarrow.

This invention also provides the instant method, wherein SDF-1production is inhibited by administration of an anti-SDF-1 or anti-CXCR4monoclonal antibody to the subject.

This invention also provides a method for treating a cancer in a subjectcomprising administering to the subject a monoclonal antibody directedagainst an epitope of a specific chemokine produced by proliferatingcells associated with the cancer so as to reduce trafficking ofendothelial progenitor cells to such proliferating cells and therebytreat the cancer in the subject.

This invention also provides a method for treating a cancer in a subjectcomprising administering to the subject a monoclonal antibody directedagainst an epitope of a specific receptor located on an endothelialprogenitor cell, for a chemokine produced by proliferating cellsassociated with the cancer, so as to reduce trafficking of theendothelial progenitor cell to such proliferating cells and therebytreat the cancer in the subject.

This invention also provides a method for treating a tumor in a subjectcomprising administering to the subject an antagonist to a specificreceptor on an endothelial progenitor cell so as to reduce theprogenitor cell's ability to induce vasculogenesis in the subject'stumor and thereby treat the tumor.

This invention also provides a method for treating a tumor in a subjectcomprising administering to the subject an antagonist to a specificreceptor on an endothelial progenitor cell so as to reduce theprogenitor cell's ability to induce angiogenesis in the subject's tumorand thereby treat the tumor.

This invention also provides the instant method, wherein the receptor isa CD117 receptor.

This invention also provides a method for expressing a gene of interestin an endothelial progenitor cell or a mast progenitor cell whichcomprises inserting into the cell a vector comprising a promotercontaining a GATA-2 motif and the gene of interest.

This invention also provides the instant method, wherein the vector isinserted into the cell by transfection.

This invention also provides the instant method, wherein the promoter isa preproendothelin-1 promoter.

This invention also provides the instant method, wherein the promoter isof mammalian origin.

This invention also provides the instant method, wherein the promoter isof human origin.

This invention provides a composition comprising an amount of amonoclonal antibody directed against an epitope of a specific chemokineproduced by a cancer effective to reduce trafficking of endothelialprogenitor cells to the cancer, and a pharmaceutically acceptablecarrier.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is treated by the expression of a GATA-2activated gene product in the subject comprising:

-   -   (a) removing stem cells from a location within the subject;    -   (b) recovering endothelial progenitor cells from the stem cells        removed in step (a);    -   (c) recovering those endothelial progenitor cells recovered in        step (b) that express GATA-2;    -   (d) inducing the cells recovered in step (c) as expressing        GATA-2 to express a GATA-2 activated gene product; and    -   (e) introducing the cells expressing a GATA-2 activated gene        product from step (d) into a different location in the subject        such as to treat the abnormality.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is treated by the expression of a GATA-2activated gene product in the subject comprising:

-   -   (a) removing stem cells from a location within the subject;    -   (b) recovering mast progenitor cells from the stem cells removed        in step (a);    -   (c) recovering those mast progenitor cells recovered in step (b)        that express GATA-2;    -   (d) inducing the cells recovered in step (c) as expressing        GATA-2 to express a GATA-2 activated gene product; and    -   (e) introducing the cells expressing a GATA-2 activated gene        product from step (d) into a different location in the subject        such as to treat the abnormality

This invention provides the instant method, wherein the abnormality isischemia-damaged tissue.

This invention provides the instant method, wherein the gene product isproendothelin.

This invention provides the instant method, wherein the gene product isendothelin.

This invention provides the a method of improving myocardial function ina subject that has suffered a myocardial infarct comprising:

-   -   (a) removing stem cells from a location in the subject;    -   (b) recovering cells that express CD117 from the stem cells; and    -   (c) introducing the recovered cells into a different location in        the subject such that the cells improve myocardial function in        the subject.

This invention provides the instant methods, wherein the subject is ofmammalian origin.

This invention provides the instant method, wherein the mammal is ofhuman origin.

This invention also provides a method of stimulating vasculogenesis inischemia-damaged tissue in a subject comprising:

-   -   (a) obtaining allogeneic stem cells;    -   (b) recovering endothelial progenitor cells from the stem cells        removed in step (a); and    -   (c) introducing the endothelial progenitor cells recovered in        step (b) into the subject such that the endothelial progenitor        cells stimulate vasculogenesis in the subject's ischemia-damaged        tissue.

This invention provides the instant method, wherein the allogeneic stemcells are obtained from embryonic, fetal or cord blood sources.

This invention provides a method of stimulating angiogenesis inischemia-damaged tissue in a subject comprising:

-   -   (a) obtaining allogeneic stem cells;    -   (b) recovering endothelial progenitor cells in the stem cells        removed in step (a); and    -   (c) introducing the endothelial progenitor cells recovered in        step (b) into the subject such that the endothelial progenitor.        cells stimulate angiogenesis in the subject's ischemia-damaged        tissue.

This invention provides the instant method, wherein the allogeneic stemcells are obtained from embryonic, fetal or cord blood sources.

This invention also provides a method of improving myocardial functionin a subject that has suffered a myocardial infarct comprising injectingG-CSF into the subject in order to mobilize endothelial progenitorcells.

This invention also provides a method of improving myocardial functionin a subject that has suffered a myocardial infarct comprising injectinganti-CXCR4 antibody into the subject.

This invention also provides the instant method further comprisingintroducing endothelial progenitor cells into the subject.

This invention also provides the instant method further comprisingintroducing G-CSF into the subject in order to mobilize endothelialprogenitor cells.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. G-CSF Mobilizes Two Human Bone Marrow-Derived PopulationsExpressing VEGF Receptors: One. With Characteristics Of MatureEndothelial Cells And A Second With Characteristics Of EmbryonicAngioblasts.

A-D depicts four-parameter flow cytometric phenotype characterization ofG-CSF mobilized bone marrow-derived cells removed by leukopharesis froma representative human donor adult (25). Only live cells were analyzed,as defined by 7-AAD staining. For each marker used, shaded areasrepresent background log fluorescence relative to isotype controlantibody.

-   A. Following immunoselection of mononuclear cells (25), >95% of live    cells express CD34.-   B. The CD34+CD117^(dim) subset contains a population with phenotypic    characteristics of mature, vascular endothelium.-   C. The CD34+CD117^(bright) subset contains a population expressing    markers characteristic of primitive hemangioblasts arising during    waves of murine and human embryogenesis.-   D. CD34+CD117^(bright) cells co-expressing GATA-2 and GATA-3 also    express AC133, another marker which defines hematopoietic cells with    angioblast potential.

FIG. 2. Bone Marrow-Derived Angioblasts (BA) Have Greater ProliferativeActivity In Response To Both VEGF And Ischemic Serum Than BoneMarrow-Derived Endothelial Cells (BMEC).

Depicted is the response of single-donor CD34-positive human cellssorted by a fluorescent GATA-2 mAb and cultured for 96 hours in RPMIwith 20% normal rat serum, ischemic rat serum or 20 ng/ml VEGF. Thenumbers of CD117^(bright)GATA-2^(pos) and CD117^(dim) GATA-2^(neg) cellswere quantitated by both [³H] thymidine uptake and by flow cytometry.

-   A. In comparison to culture in normal serum, the proliferative    responses to either VEGF or ischemic serum were significantly higher    for CD117^(bright)-GATA-2^(pos) BA relative to    CD117^(d)-GATA-2^(neg)BMEC from the same donor (both p<0.01).-   B. The population expanded by culture with either VEGF or ischemic    serum and characterized by multiparameter flow cytometric analysis    as CD117^(bright)GATA-2^(pos) consisted of large blast cells, as    demonstrated by high forward scatter (fsc).-   C. The expanded population of CD117^(bright)GATA-2^(pos) cells did    not demonstrate increased surface expression of mature endothelial    cell markers after culture with VEGF in comparison to culture with    normal medium, indicating blast proliferation without    differentiation.

FIG. 3. Highly Purified Human Bone Marrow-Derived CD34 CellsDifferentiate Into Endothelial Cells After in Vitro Culture.

Culture of highly-purified CD34+ human cells for 7 days in endothelialgrowth medium results in outgrowth of cells with morphologic andcharacteristic features of mature endothelial cell monolayers. Themajority of the monolayers (>90%) demonstrate:

-   A. Exuberant cobblestone pattern of cellular proliferation and    growth;-   B. Uniform uptake of DiI-labeled acetylated LDL;-   C. CD34 expression, as measured by immunofluorescence using a    fluorescein-conjugated mAb;-   D. Factor VIII expression, as measured by immunoperoxidase using a    biotin-conjugated mAb; and-   E. Expression of eNOS, determined by in situ hybridization using a    specific probe.

FIG. 4. In vivo migratory and proliferative characteristics of bonemarrow- and peripheral vasculature-derived human cells after inductionof myocardial ischemia.

-   A-C. Intravenous injection of 2×10⁶ DiI-labeled human CD34-enriched    cells (>95% CD34 purity), CD34-negative cells (<5% CD34 purity), or    saphenous vein endothelial cells (SVEC), into nude rats after    coronary artery ligation and infarction. Each human cellular    population caused a similar degree of infiltration in infarcted rat    myocardium at 48 hours.-   D. A sham procedure, with no human cells found in the non-infarcted    rat heart.-   E. Measurement of human GATA-2 mRNA expression in the bone marrow    and heart of infarcted rats receiving either CD34-positive cells    (>95% CD34 purity), CD34-negative cells (<5% CD34 purity),    normalized for total human RNA measured by GAPDH expression. GATA-2    mRNA in ischemic tissue is expressed as the fold increase above that    present under the same experimental condition in the absence of    ischemia. Bone marrow from ischemic rats receiving either CD34+ or    CD34-cells contained similar levels of human GATA-2 mRNA, and showed    a similar fold induction in GATA-2 mRNA expression after ischemia.    In contrast, ischemic hearts of rats receiving CD34+ cells contained    much higher levels of human GATA-2 mRNA than those receiving    CD34-cells. Moreover, the degree of increase in GATA-2 mRNA    expression after infarction was 2.6-fold higher for hearts    infiltrated by CD34+ cells compared with CD34-cells, indicating that    GATA-2+ cells within the CD34+ fraction selectively traffic to    ischemic myocardium.-   F. Consecutive sections of a blood vessel within the infarct bed of    a nude rat two weeks after injection of human CD34+ cells. The    vessel incorporates human endothelial cells, as defined by    co-expression of DiI, HLA class I as measured by immunofluorescence    using a fluorescein-conjugated mAb, and factor VIII, as measured by    immunoperoxidase using a biotin-conjugated mAb.

FIG. 5. Injection of G-CSF Mobilized Human CD34+Cells Into Rats WithAcute Infarction Improves Myocardial Function.

A-D compares the functional effects of injecting 2×10⁶ G-CSF mobilizedhuman CD34+(>95% purity) cells, CD34− (<5% purity) cells, peripheralsaphenous vein cells, or saline, into infarcted rat myocardium.

-   A. Although left ventricular ejection fraction (LVEF) was severely    depressed in each group of recipients after LAD ligation, only    injection of G-CSF mobilized adult human CD34+ cells was accompanied    by significant, and sustained, LVEF recovery (p<0.001). LVEF    recovery was calculated as the mean % improvement between LVEF after    LAD ligation and pre-infarct LVEF.-   B. Similarly, although left ventricular end-systolic area (LVAs) was    markedly increased in each group of recipients after LAD ligation,    only injection of G-CSF mobilized adult human CD34+ cells was    accompanied by significant, and sustained, reduction in LVAs    (p<0.001). Reduction in LVAs was calculated as the mean %    improvement between LVAs after LAD ligation and pre-infarct LVAs.-   C. Representative echocardiographic examples from each group are    shown. At 48 hours after LAD ligation, diastolic function is    severely compromised in each rat. At two weeks after injection,    diastolic function is improved only in the rat receiving CD34+    cells. This effect persists at 15 weeks.-   D. At 15 weeks post-infarction, rats injected with CD34+ cells    demonstrated significantly less reduction in mean cardiac index    relative to normal rats than each of the other groups (p<0.001).

FIG. 6. Injection Of G-CSF Mobilized Human CD34+Cells Into Rats WithAcute Infarction Induces Neo-Angiogenesis And Modifies The Process OfMyocardian Remodeling.

A-D depicts infarcted rat myocardium at two weeks post-LAD ligation fromrepresentative experimental and control animals stained with eitherhematoxylin and eosin (A,B) or immunoperoxidase following binding ofanti-factor VIII mAb (C,D). E,F depicts Mason trichrome stain ofinfarcted rat myocardium from representative control and experimentalanimals at 15 weeks post-LAD ligation. G depicts between-groupdifferences in % scar/normal left ventricular tissue at 15 weeks.

-   A. Infarct zone of rat injected with human CD34+ cells demonstrates    significant increase in microvascularity and cellularity of    granulation tissue, numerous capillaries (arrowheads), feeding    vessels (arrow), and decrease in matrix deposition and fibrosis    (×200).-   B. In contrast, infarct zone of control rat injected with saline    shows a myocardial scar composed of paucicellular, dense fibrous    tissue (arrows) (×200).-   C. Ischemic myocardium of rat injected with human CD34+ cells    demonstrates numerous factor VIII-positive interstitial angioblasts    (arrows), and diffuse increase in factor VIII-positive capillaries    (arrowheads) (×400).-   D. Ischemic myocardium of rat injected with saline does not contain    factor VIII-positive angioblasts (arrows), and demonstrates only    focal areas of granulation tissue with factor VIII positive    vascularity (arrowheads) (×400).-   E. Trichrome stain of rat myocardium at 15 weeks post-infarction in    rat injected with saline (×25). The collagen rich myocardial scar in    the anterior wall of the left ventricle (ant.) stains blue and    viable myocardium stains red. Focal islands of collagen deposition    (blue) are also present in the posterior wall of the left ventricle    (post). There is extensive loss of anterior wall myocardial mass,    with collagen deposition and scar formation extending almost through    the entire left ventricular wall thickness, causing aneurysmal    dilatation and typical EKG abnormalities (persistent ST segment    elevation).-   F. In contrast, trichrome stain of rat myocardium at 15 weeks    post-infarction in rat receiving highly purified CD34+ cells (×25)    demonstrates significantly reduced infarct zone size together with    increased mass of viable myocardium within the anterior wall (ant.)    and normal EKG. Numerous vessels are evident at the junction of the    infarct zone and viable myocardium. There is no focal collagen    deposition in the left ventricular posterior wall (post).-   G. Rats receiving CD34+ cells had a significant reduction in mean    size of scar tissue relative to normal left ventricular myocardium    compared with each of the other groups (p<0.01). Infarct size,    involving both epicardial and endocardial regions, was measured with    a planimeter digital image analyzer and expressed as a percentage of    the total ventricular circumference at a given slice. For each    animal, final infarct size was calculated as the average of 10-15    slices.

FIG. 7. Human Adult Bone Marrow-Derived Endothelial Precursor CellsInfiltrate Ischemic Myocardium, Inducing Infarct Bed Neoangiogenesis AndPreventing Collagen Deposition.

-   A. Four-parameter flow cytometric phenotypic characterization of    G-CSF mobilized bone marrow-derived cells removed by leukopheresis    from a representative human donor adult. Only live cells were    analyzed, as defined by 7-AAD staining. For each marker used, shaded    areas represent background log fluorescence relative to isotype    control antibody. The CD34+CD117^(bright) subset contains a    population expressing markers characteristic of primitive    haemangioblasts arising during waves of murine and human    embryogenesis, but not markers of mature endothelium. These cells    also express CXC chemokine receptors.-   B. DiI-labeled human CD34-enriched cells (>98% CD34 purity) injected    intravenously into nude rats infiltrate rat myocardium after    coronary artery ligation and infarction but not after sham operation    at 48 hours.-   C. The myocardial infarct bed at two weeks post-LAD ligation from    representative rats receiving 2.0×10⁶ G-CSF mobilized human bone    marrow-derived cells at 2%, 40%, or 98% CD34+ purity, and stained    with either Masson's trichrome or immunoperoxidase. The infarct    zones of rats receiving either 2% or 40% pure CD34+ cells show    myocardial scars composed of paucicellular, dense fibrous tissue    stained blue (×400). In contrast, the infarct zone of the rat    injected with 98% pure human CD34+ cells demonstrates significant    increase in microvascularity and cellularity of granulation tissue,    numerous capillaries, and minimal matrix deposition and fibrosis    (×400). Moreover, immunoperoxidase staining following binding of    anti-factor VIII mAb shows that the infarct bed of the rat injected    with 98% pure CD34+ cells demonstrates markedly increased numbers of    factor VIII-positive capillaries, which are not seen in either of    the other animals (×400).

FIG. 8. Migration Of Human Bone Marrow-Derived Endothelial PrecursorCells To The Site Of Infarction Is Dependent On Interactions BetweenCXCR1/2 And IL-8/Gro-Alpha Induced By Myocardial Ischemia.

-   A,B. Time-dependent increase in rat myocardial IL-8 and Gro-alpha    mRNA expression relative to GAPDH from rats undergoing LAD ligation.-   C. IL-8, Gro-alpha, and GAPDH mRNA expression at baseline, 12 hours    and 48 hours after LAD ligation from a representative animal.-   D. Time-dependent measurement of rat IL-8/Gro-alpha protein in serum    of rats undergoing LAD ligation. Migration of CD34+ human bone    marrow-derived cells to ischemic rat myocardium is inhibited by mAbs    against either rat IL-8 or the IL-8/Gro chemokine family receptors    CXCR1 and CXCR2 (all p<0.01), but not against VEGF or its receptor    Flk-1 (results are expressed as mean±sem of three separate    experiments).-   E. Co-administration of blocking mAbs against either IL-8 and    Gro-alpha, or against the surface receptors for these pro-angiogenic    chemokines, CXCR-1 and CXCR-2, reduced myocardial trafficking of    human angioblasts by 40-60% relative to control antibodies (p<0.01).

FIG. 9. CXC Chemokines Directly Induce Chemotaxis Of Bone Marrow-DerivedHuman CD34+Cells To Rat Myocardium.

A and B depict results of in vitro chemotaxis of 98% pure human CD34+cells to various conditions using a 48-well chemotaxis chamber (NeuroProbe, MD). Chemotaxis is defined as the number of migrating cells perhigh power field (hpf) after examination of 10 hpf per condition tested.

-   A. IL-8 induces chemotaxis in a dose-dependent manner (results are    expressed as mean±sem of three separate experiments).-   B. Chemotaxis is increased in response to IL-8 and SDF-1 alpha/beta,    but not VEGF or SCF.-   C. Representative fluorescence microscopy demonstrating increased    infiltration of intravenously-injected DiI-labeled human CD34+ cells    (98% purity) into rat heart after intracardiac injection with IL-8    compared with saline injection.-   D. Intracardiac injection of IL-8 at 1 mg/ml significantly increases    in vivo chemotaxis of DiI-labeled human CD34+ cells (98% purity)    into rat heart in comparison with injection of saline, VEGF or stem    cell factor (SCF), p<0.01 (results are expressed as mean±sem of    three separate experiments).

FIG. 10. Blocking CXCR4/SDF-1 Interactions Redirects IntravenouslyInjected Human CD34+ Angioblasts From Bone Marrow To IschemicMyocardium.

-   A. Depicted is the response of single-donor CD34-positive human    cells cultured for 96 hours in RPMI with 20% normal rat serum,    ischemic rat serum or 20 ng/ml VEGF. The numbers of    CD117^(bright)GATA-2^(pos) cells were quantitated by both [³H]    thymidine uptake and by flow cytometry. Ischemic serum induced a    greater proliferative response of CD117^(bright)GATA-2^(pos) cells    compared with each of the other conditions (both p<0.01).

B. The proportion of human CD34+ cells in rat bone marrow 2-14 daysafter intravenous injection is significantly increased after ischemiainduced by LAD ligation (results are expressed as mean±sem of bonemarrow studies in three animals at each time point).

-   C,D. Effects of mAbs against CXCR4, SDF-1 or anti-CD34 on    trafficking of human CD34+ cells to rat bone marrow and myocardium    following LAD ligation. Co-administration of anti-CXCR4 or    anti-SDF-1 significantly reduced trafficking of 98% pure CD34+ cells    to rat bone marrow at 48 hours and increased trafficking to ischemic    myocardium (results are expressed as mean±sem of bone marrow and    cardiac studies performed in three LAD-ligated animals at 48 hours    after injection).

FIG. 11. Redirected Trafficking Of Human CD34+ Angioblasts To The SiteOf Infarction Prevents Remodeling And Improves Myocardial Function.

-   A,B. The effects of human CD34+ cells on reduction in LVAs (A) and    improvement in LVEF (B) after myocardial infarction. Whereas    injection of 2.0×10⁶ human cells containing 98% CD34+ purity    significantly improved LVEF and reduced LVAs (both p<0.01),    injection of 2.0×10⁶ human cells containing 2% and 40% CD34+ purity    did not have any effect on these parameters in comparison to animals    receiving saline. However, co-administration of anti-CXCR4 together    with 40% pure CD34+ cells significantly improved LVEF and reduced    LVAs (both p<0.01), to levels approaching use of cells with 98%    purity.-   C. Sections of rat hearts stained with Masson's trichrome at 15    weeks after LAD ligation and injection of 2.0×10⁶ human cells    containing 2%, 40%, or 98% CD34+ purity. Hearts of rats receiving 2%    and 40% pure CD34+ cells had greater loss of anterior wall mass,    collagen deposition (blue), and septal hypertrophy compared with    hearts of rats receiving 98% pure CD34+ cells. Co-administration of    anti-CXCR4 mAb together with 40% pure CD34+ cells increased left    ventricular wall mass and reduced collagen deposition.-   D. Shows the mean proportion of scar/normal left ventricular    myocardium in rats receiving >98% pure CD34+ cells or 40% pure CD34+    cells together with anti-CXCR4 mAb is significantly reduced in    comparison to rats receiving 2% and 40% pure CD34+ cells (p<0.01)    (results are expressed as mean t sem of three separate experiments).

FIG. 12. Culture of CD34+CD117^(bright) angioblasts with serum fromLAD-ligated rats increases surface expression of CCR1 and CCR2, whilesurface expression of CCR3 and CCR5 remains unchanged.

FIG. 13. Infarcted myocardium demonstrate a time-dependent increase inmRNA expression of several CCR-binding chemokines.

FIG. 14. Co-administration of blocking mAbs against MCP-1, MCP-3, andRANTES, or against eotaxin, reduced myocardial trafficking of humanangioblasts by 40-60% relative to control antibodies (p<0.01).

FIG. 15. Intracardiac injection of eotaxin into non-infarcted heartsinduced 1.5-1.7 fold increase in CD34+ angioblast trafficking whereasinjection of the growth factors VEGF and stem cell factor had no effecton chemotaxis despite increasing angioblast proliferation (not shown).

DETAILED DESCRIPTION OF THE INVENTION

As used herein, and unless stated otherwise, each of the following termsshall have the definition set forth below.

As used herein, “BMEC” is defined as bone marrow-derived endothelialcells.

As used herein, vasculogenesis is defined as the creation of new bloodvessels from cells that are “pre-blood” cells such as bonemarrow-derived endothelial cell precursors.

As used herein, mobilization is defined as inducing bone marrow-derivedendothelial cell precursors to leave the bone marrow and enter theperipheral circulation. One of skill is aware that mobilized stem cellsmay be removed from the body by leukopheresis.

As used herein, ischemia is defined as inadequate blood supply(circulation) to a local area due to blockage of the blood vessels tothe area.

As used herein, cytokine is defined as a factor that causes cells togrow or activate.

As used herein, chemokine is defined as a factor that causes cells tomove to a different area within the body.

As used herein, ischemic heart disease is defined as any condition inwhich blood supply to the heart is decreased.

As used herein, “angiogenesis” is defined as the creation of bloodvessels from pre-existing blood vessel cells.

As used herein, ischemic heart disease is defined as any condition inwhich blood supply to the heart is decreased.

As used herein, “VEGF” is defined as vascular endothelial growth factor.“VEGF-R” is defined as vascular endothelial growth factor receptor.“FGF” is defined as fibroblast growth factor. “IGF” is defined asInsulin-like growth factor. “SCF” is defined as stem cell factor.“G-CSF” is defined as granulocyte colony stimulating factor. “M-CSF” isdefined as macrophage colony stimulating factor. “GM-CSF” is defined asgranulocyte-macrophage colony stimulating factor. “MCP” is defined asmonocyte chemoattractant protein.

As used herein, “CXC” chemokine refers to the structure of thechemokine. Each “C” represents a cysteine and “X” represents any aminoacid.

As used herein, “CC” chemokine refers to the structure of the chemokine.Each “C” represents a cysteine.

As used herein, “recovered” means detecting and obtaining a cell basedon the recoverable cell being a cell that binds a detectably labeledantibody directed against a specific marker on a cell including, but notlimited to, CD117, GATA-2, GATA-3, and CD34.

As described herein, the chemokine administered to the subject could bein the protein form or nucleic acid form.

This invention provides a method of stimulating vasculogenesis inischemia-damaged tissue of a subject comprising:

-   -   (a) removing stem cells from a location within the subject;    -   (b) recovering endothelial progenitor cells from the stem cells        removed in step (a); and    -   (c) introducing the endothelial progenitor cells from step (b)        into a different location within the subject such that the        endothelial progenitor cells stimulate vasculogenesis in the        subject's ischemia-damaged tissue.

In a further embodiment the endothelial progenitors are frozen for aperiod of time in between steps (b) and (c). In one embodiment theischemia-damaged tissue is myocardium. In another embodiment theischemia-damaged tissue is nervous system tissue.

In one embodiment the endothelial progenitors are expanded by contactingthe endothelial progenitors with a growth factor subsequent to step (b),but before step (c). In a further embodiment the growth factor is acytokine. In further embodiments the cytokine is VEGF, FGF, G-CSF, IGF,M-CSF, or GM-CSF. In another embodiment the growth factor is achemokine. In a further embodiment the chemokine is Interleukin-8. Inone embodiment the endothelial progenitors are separated from other stemcells before expansion. In a further embodiment the endothelialprogenitors are frozen for a period of time after expansion but beforestep (c).

In one embodiment step (a) occurs prior to the subject sufferingischemia-damaged tissue and wherein step (c) occurs after the subjecthas suffered ischemia-damaged tissue.

In one embodiment the stem cells are removed directly from the subject'sbone marrow. In a further embodiment the stem cells are removed byaspiration from the subject's bone marrow. In one embodiment the stemcells are removed from the subject by a method comprising:

-   -   a) introducing a growth factor into the subject to mobilize the        stem cells into the subject's blood; and    -   b) subsequently removing a sample of blood containing stem cells        from the subject.

In a further embodiment the growth factor is introduced subcutaneously,orally, intravenously or intramuscularly.

In one embodiment the growth factor is a chemokine that inducesmobilization. In a further embodiment the chemokine is Interleukin-8. Inone embodiment the growth factor is a cytokine. In a further embodimentthe cytokine is G-CSF, M-CSF, or GM-CSF.

This invention also provides the instant method, wherein the endothelialprogenitor cells are recovered based upon their expression of CD117.

This invention also provides the instant method, wherein the endothelialprogenitor cells are recovered based upon their expression of a GATA-2activated gene product. In one embodiment the gene product is selectedfrom the following group: preproendothelin-1, big endothelin,endothelin-1.

In one embodiment the endothelial progenitors express GATA-2, and theendothelial progenitors are recovered as such by detection ofintracellular GATA-2 expression or GATA-2 activity in those cells.

In one embodiment the subject has suffered or is suffering from one ormore of the following: myocardial infarction, chronic heart failure,ischemic heart disease, coronary artery disease, diabetic heart disease,hemorrhagic stroke, thrombotic stroke, embolic stroke, limb ischemia oranother disease in which tissue is rendered ischemic.

In one embodiment the endothelial progenitors are introduced into thesubject by injection directly into the peripheral circulation, heartmuscle, left ventricle, right ventricle, coronary artery, cerebro-spinalfluid, neural tissue, ischemic tissue or post-ischemic tissue.

In one embodiment the method further comprises administering to thesubject one or more of the following: an inhibitor of PlasminogenActivator Inhibitor, Angiotensin Converting Enzyme Inhibitor or a betablocker, wherein such administration occurs prior to, concomitant with,or following step (c).

This invention also provides a method of stimulating angiogenesis inperi-infarct tissue in a subject comprising:

-   -   (a) removing stem cells from a location within a subject;    -   (b) recovering endothelial progenitor cells from the stem cells        removed in step (a);    -   (c) expanding the endothelial progenitor cells recovered in        step (b) by contacting the progenitor cells with a growth        factor; and    -   (d) introducing the expanded endothelial progenitor cells from        step (c) into a different location in the subject such that the        endothelial progenitor cells stimulate angiogenesis in        peri-infarct tissue in the subject.

This invention also provides a method of selectively increasing thetrafficking of endothelial progenitor cells to ischemia-damaged tissuein a subject comprising:

-   -   (a) administering endothelial progenitor cells to a subject; and    -   (b) administering a chemokine to the subject so as to thereby        attract the endothelial progenitor cells to the ischemia-damaged        tissue.

In one embodiment the chemokine is administered to the subject prior toadministering the endothelial progenitors. In an alternative embodimentthe chemokine is administered to the subject concurrently with theendothelial progenitors. In an alternative embodiment the chemokine isadministered to the subject after administering the endothelialprogenitors. In one embodiment the chemokine is a CXC chemokine. In afurther embodiment the CXC chemokine is selected from the groupconsisting of Interleukin-8, Gro-Alpha, or Stromal-Derived Factor-1. Inone embodiment the chemokine is a CC chemokine. In a further embodimentthe CC chemokine is selected from the group consisting of RANTES,EOTAXIN, MCP-1, MCP-2, MCP-3, or MCP-4.

In one embodiment the chemokine is administered to the subject byinjection into peripheral circulation, heart muscle, left ventricle,right ventricle, coronary arteries, cerebro-spinal fluid, neural tissue,ischemic tissue or post-ischemic tissue.

This invention also provides a method of increasing trafficking ofendothelial progenitor cells to ischemia-damaged tissue in a subjectcomprising inhibiting any interaction between Stromal-Derived Factor-1and CXCR4.

In one embodiment the interaction between Stromal-Derived Factor-1(SDF-1) and CXCR4 is inhibited by administration of an anti-SDF-1 or ananti-CXCR4 monoclonal antibody to the subject. In one embodiment theinstant method further comprises administering to the subject ACEinhibitor, AT-receptor blocker, or beta blocker ng enzyme inhibitor, anAT₁-receptor blocker, or a beta blocker.

This invention also provides a method of reducing trafficking ofendothelial progenitor cells to bone marrow in a subject comprisinginhibiting production of Stromal-Derived Factor-1 in the subject's bonemarrow. In one embodiment the SDF-1 production is inhibited byadministration of an anti-SDF-1 or anti-CXCR4 monoclonal antibody to thesubject.

This invention also provides a method for treating a cancer in a subjectcomprising administering to the subject a monoclonal antibody directedagainst an epitope of a specific chemokine produced by proliferatingcells associated with the cancer so as to reduce trafficking ofendothelial progenitor cells to such proliferating cells and therebytreat the cancer in the subject.

This invention also provides a method for treating a cancer in a subjectcomprising administering to the subject a monoclonal antibody directedagainst an epitope of a specific receptor located on an endothelialprogenitor cell, for a chemokine produced by proliferating cellsassociated with the cancer, so as to reduce trafficking of theendothelial progenitor cell to such proliferating cells and therebytreat the cancer in the subject.

This invention also provides a method for treating a tumor in a subjectcomprising administering to the subject an antagonist to a specificreceptor on an endothelial progenitor cell so as to reduce theprogenitor cell's ability to induce vasculogenesis in the subject'stumor and thereby treat the tumor.

This invention also provides a method for treating a tumor in a subjectcomprising administering to the subject an antagonist to a specificreceptor on an endothelial progenitor cell so as to reduce theprogenitor cell's ability to induce angiogenesis in the subject's tumorand thereby treat the tumor.

This invention also provides a method for expressing a gene of interestin an endothelial progenitor cell or a mast progenitor cell whichcomprises inserting into the cell a vector comprising a promotercontaining a GATA-2 motif and the gene of interest.

This invention also provides the instant method, wherein the vector isinserted into the cell by transfection.

This invention also provides the instant method, wherein the promoter isa preproendothelin-1 promoter.

This invention also provides the instant method, wherein the promoter isof mammalian origin.

This invention also provides the instant method, wherein the promoter isof human origin.

This invention provides a composition comprising an amount of amonoclonal antibody directed against an epitope of a specific chemokineproduced by a cancer effective to reduce trafficking of endothelialprogenitor cells to the cancer, and a pharmaceutically acceptablecarrier.

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is treated by the expression of a GATA-2activated gene product in the subject comprising:

-   -   (a) removing stem cells from a location within the subject;    -   (b) recovering endothelial progenitor cells from the stem cells        removed in step (a);    -   (c) recovering those endothelial progenitor cells recovered in        step (b) that express GATA-2;    -   (d) inducing the cells recovered in step (c) as expressing        GATA-2 to express a GATA-2 activated gene product; and    -   (e) introducing the cells expressing a GATA-2 activated gene        product from step (d) into a different location in the subject        such as to treat the abnormality.

In one embodiment the abnormality is ischemia-damaged tissue. In oneembodiment the gene product is proendothelin. In one embodiment the geneproduct is endothelin. In one embodiment the subject is a mammal. In afurther embodiment the mammal is a human

This invention provides a method of treating an abnormality in a subjectwherein the abnormality is treated by the expression of a GATA-2activated gene product in the subject comprising:

-   -   (a) removing stem cells from a location within the subject;    -   (b) recovering mast progenitor cells from the stem cells removed        in step (a);    -   (c) recovering those mast progenitor cells recovered in step (b)        that express GATA-2;    -   (d) inducing the cells recovered in step (c) as expressing        GATA-2 to express a GATA-2 activated gene product; and    -   (e) introducing the cells expressing a GATA-2 activated gene        product from step (d) into a different location in the subject        such as to treat the abnormality

In one embodiment the abnormality is ischemia-damaged tissue. In oneembodiment the gene product is proendothelin. In one embodiment the geneproduct is endothelin. In one embodiment the subject is a mammal. In afurther embodiment the mammal is a human

This invention provides the a method of improving myocardial function ina subject that has suffered a myocardial infarct comprising:

-   -   (a) removing stem cells from a location in the subject;    -   (b) recovering cells that express CD117 from the stem cells; and    -   (c) introducing the recovered cells into a different location in        the subject such that the cells improve myocardial function in        the subject.

In one embodiment the subject is a mammal. In a further embodiment themammal is a human.

This invention also provides a method of stimulating vasculogenesis inischemia-damaged tissue in a subject comprising:

-   -   (a) obtaining allogeneic stem cells;    -   (b) recovering endothelial progenitor cells from the stem cells        removed in step (a); and    -   (c) introducing the endothelial progenitor cells recovered in        step (b) into the subject such that the endothelial progenitor        cells stimulate vasculogenesis in the subject's ischemia-damaged        tissue.

In alternative embodiments the allogeneic stem cells are removed fromembryonic, fetal or cord blood sources.

This invention provides a method of stimulating angiogenesis inischemia-damaged tissue in a subject comprising:

-   -   (a) obtaining allogeneic stem cells;    -   (b) recovering endothelial progenitor cells in the stem cells        removed in step (a); and    -   (c) introducing the endothelial progenitor cells recovered in        step (b) into the subject such that the endothelial progenitor        cells stimulate angiogenesis in the subject's ischemia-damaged        tissue.

In alternative embodiments the allogeneic stem cells are removed fromembryonic, fetal or cord blood sources.

This invention also provides a method of improving myocardial functionin a subject that has suffered a myocardial infarct comprising injectingG-CSF into the subject in order to mobilize endothelial progenitorcells.

This invention also provides a method of improving myocardial functionin a subject that has suffered a myocardial infarct comprising injectinganti-CXCR4 antibody into the subject. In one embodiment the methodfurther comprises introducing endothelial progenitors into the subject.In one embodiment the method further comprises introducing G-CSF intothe subject in order to mobilize endothelial progenitors.

The present invention provides a method of selectively increasing thetrafficking of human marrow-derived endothelial cell precursors to thesite of tissue damaged by ischemic injury which comprises administeringchemokines to the subject so as to thereby attract endothelial cellprecursors to the ischemic tissue. In an embodiment of this inventionthe ischemic tissue is myocardium. In an embodiment of this inventionthe ischemic tissue is neural tissue. In an embodiment of this inventionthe chemokine is administered to the subject by injection intoperipheral circulation, heart muscle, left ventricle, right ventricle,coronary arteries, spinal fluid, neural tissue, or other site ofischemia. In an embodiment of this invention the chemokine is a CXCchemokine. In an embodiment of this invention the CXC chemokine isselected from the group consisting of Interleukin-8 (IL-8), Gro-Alpha,and Stromal Derived Factor-1 (SDF-1). In an embodiment of this inventionthe chemokine is a CC chemokine. In an embodiment of this invention theCC chemokine is selected from the group consisting of MCP-1, MCP-2,MCP-3, MCP-4, RANTES, and EOTAXIN.

This invention will be better understood by reference to theExperimental Details which follow, but those skilled in the art willreadily appreciate that the specific experiments detailed are onlyillustrative of the invention as described more fully in the claimswhich follow thereafter.

Experimental Details

First Series of Experiments

Experimental Procedures and Results

1. Mobilization and Identification of Bone Marrow-Derived Cells

Following G-CSF mobilization, 60-80% of highly purified human CD34 cells(>90% positive) co-expressed the stem cell factor receptor CD117, FIG. 1a, of which 15-25% expressed CD117 brightly and 75-85% expressed CD117dimly. By quadruple parameter analysis, two populations of CD34 cellswere recovered which expressed VEGFR-2 (Flk-1), one accounting for20-30% of CD117^(dim) cells and expressing high levels of VEGFR-2, and asecond accounting for 10-15% of CD117^(bright) cells and expressinglower levels of VEGFR-2, FIG. 1 b. The VEGFR-2 positive cells within theCD34+CD117^(dim) population, but not those within theCD34+CD117^(bright) subset, displayed phenotypic characteristics ofmature, vascular endothelium, including high level expression of Tie-2,ecNOS, vWF, E-selectin (CD62E), and ICAM (CD54). In contrast, as shownin FIG. 1 c, the VEGFR-2 positive cells within the CD34+CD117^(bright)subset, but not those within the CD34+CD117^(dim) subset, expressedmarkers characteristic of primitive hemangioblasts arising during wavesof murine and human embryogenesis, including GATA-2, GATA-3, and lowlevels of Tie-2. Moreover, CD117^(bright) cells which co-expressedGATA-2 and GATA-3 were also strongly AC133 positive, another markerwhich has recently been suggested to define a hematopoietic populationwith angioblast potential (2), FIG. 1 d. However, since AC133 expressionwas also detected on a subset of CD117^(dim) cells which was negativefor GATA-2 and GATA-3, we conclude that identification of an embryonicbone-marrow derived angioblast (BA) phenotype requires concomitantexpression of GATA-2, GATA-3, and CD117^(bright) in addition to AC133.Thus, G-CSF treatment mobilizes into the peripheral circulation aprominent population of mature, bone marrow-derived endothelial cells(BMEC), and a smaller bone marrow-derived population with phenotypiccharacteristics of embryonic angioblasts (BA).

2. Expansion of Bone Marrow-Derived Cells

Since the frequency of circulating endothelial cell precursors in animalmodels has been shown to be increased by either VEGF (27) or regionalischemia (10-13), we next compared the proliferative responses of BA andBMEC to VEGF and to factors in ischemic serum (28). As shown in FIG. 2a, following culture for 96 hours with either VEGF or ischemic serum,CD117^(bright)GATA-2^(pos) BA demonstrated significantly higherproliferative responses relative to CD117^(dim)GATA-2^(neg) BMEC fromthe same donor. For VEGF, BA showed 2.9-fold increase in proliferationabove baseline compared with 1.2-fold increase for BMEC, p<0.01, whilefor ischemic serum from Lew rats with myocardial infarction BA showed4.3-fold increase in proliferation above normal serum compared with1.7-fold increase for BMEC, p<0.01. Culture with either VEGF or ischemicserum greatly expanded the BA population of large blast cells, FIG. 2 b,which continued to express immature markers, including GATA-2, GATA-3,and CD117^(bright), but not markers of mature endothelial cells, FIG. 2c, indicating blast proliferation without differentiation. Followingculture of CD34-positive monolayers on fibronectin in endothelial growthmedium for 7 days (29), an exuberant cobblestone pattern ofproliferation was seen, FIG. 3 a, with the majority of the adherentmonolayers (>95%) having features characteristic of endothelial cells,FIG. 3 b-e, including uniform uptake of acetylated LDL, andco-expression of CD34, factor VIII, and eNOS. Since the BMEC populationhad low proliferative responses to VEGF or cytokines in ischemic serum,the origin of the exuberant endothelial cell outgrowth in culture ismost likely the BA population defined by surface expression for GATA-2,GATA-3, and CD117^(bright).

3. In vivo Migration of Bone Marrow-Derived CD34+ Cells to Sites ofRegional Ischemia

Next we compared the in vivo migratory and proliferative characteristicsof bone marrow- and peripheral vasculature-derived human cells afterinduction of regional ischemia. As shown in FIG. 4 a-c, intravenousinjection of 2×10⁶ DiI-labeled human CD34-positive cells (>95% CD34purity), CD34-negative cells (<5% CD34 purity), or saphenous veinendothelial cells (SVEC), into nude rats after coronary artery ligationand infarction resulted in similar degree of infiltration in ratmyocardium at 48 hours (30) The trafficking was specifically directed tothe infarct area since few DiI-labeled cells were detected in unaffectedareas of hearts with regional infarcts, not shown, and neither G-CSFmobilized CD34+ cells nor mature human endothelial cells infiltratednormal myocardium, FIG. 4 d. Although similar numbers of CD34+ and CD34−cells migrated to ischemic myocardium, the proportional increase inhuman GATA-2 mRNA expression in ischemic myocardium relative to normalmyocardium (31) was 2.6-fold greater following injection of highlyCD34-enriched cells compared with CD34− cells (p<0.001), FIG. 4 e.Moreover, blood vessels which incorporated human endothelial cells, asdefined by co-expression of DiI, HLA class I, and factor VII, could bedetected two weeks after injection of human CD34+ cells, but not afterinjection of CD34− cells or SVEc, FIG. 4 f. Together, these resultsindicate that adult bone marrow-derived human CD34+ cells contain apopulation which selectively responds to in vivo signals from sites ofregional ischemia with augmented migration, localization, andendothelial differentiation.

4. Effects of Injection of G-CSF mobilized Human CD34+ Cells intoInfarcted Rat Myocardium

We next compared the functional effects of injecting G-CSF mobilizedhuman CD34+ (>95%) cells, CD34− (<5%) cells, peripheral saphenous veincells, or saline, into infarcted rat myocardium. After LAD ligation,left ventricular function was severely depressed in each group ofrecipients, with left ventricular ejection fraction (LVEF) being reducedby means of 25-43% and left ventricular end-systolic area beingincreased by means of 44-90%, FIG. 5 a and b. Remarkably, within twoweeks of injecting G-CSF mobilized adult human CD34+ cells, LVEFrecovered by a mean of 22±6% (p<0.001), FIG. 5 a. This effect waslong-lived, and increased by the end of follow-up, 15 weeks, to 34±4%.In contrast, injection of G-CSF mobilized human CD34− cells, saphenousvein endothelial cells, or saline, had no effect on LVEF. In a parallelfashion, injection of G-CSF mobilized human CD34+ cells reduced leftventricular end-systolic area by a mean of 26±8% by 2 weeks and 37±6% by15 weeks, whereas none of the other recipient groups demonstrated sucheffect (p<0.001), FIG. 5 b. Representative echocardiographic examplesfor each group are shown in FIG. 5 c. Moreover, at 15 weekspost-infarction mean cardiac index in rats injected with CD34+ cells wasonly reduced by 26±8% relative to normal rats, whereas mean cardiacindex for each of the other groups was reduced by 48-59% (p<0.001), FIG.5 d.

Histologic examination at two weeks post-infarction (33) revealed thatinjection of CD34+ cells was accompanied by significant increase inmicrovascularity and cellularity of granulation tissue, and decrease inmatrix deposition and fibrosis within the infarct zone in comparison tocontrols, FIGS. 6 a and b. Moreover, ischemic myocardium of ratsinjected with human CD34+ cells contained significantly greater numbersof factor VIII-positive interstitial angioblasts and capillaries incomparison to ischemic myocardium of control rats, FIGS. 6 c and d.Quantitation of capillary numbers demonstrated a significant increase inneo-angiogenesis within the infarct zone of rats who received CD34+cells (mean number of factor VIII-positive capillaries per high powerfield 92±5 vs 51±4 in saline controls, p<0.01), but not within normalmyocardium (36±2 vs 37±3 capillaries per high power field). No increasein capillary numbers were observed in ischemic rat myocardiuminfiltrated with CD34− cells or SVEC. At 15 weeks post-infarction, ratsreceiving highly purified CD34+ cells demonstrated significantly reducedinfarct zone sizes together with increased mass of viable myocardiumwithin the anterior free wall compared to each of the other groups,FIGS. 6 e and f. Numerous vessels were evident at the junction of theinfarct zone and viable myocardium in tissues infiltrated with CD34+cells. Whereas collagen deposition and scar formation extended almostthrough the entire left ventricular wall thickness in controls, withaneurysmal dilatation and typical EKG abnormalities, the infarct scarextended only to 20-50% of the left ventricular wall thickness in ratsreceiving CD34+ cells. Moreover, pathological collagen deposition in thenon-infarct zone was markedly reduced in rats receiving CD34+ cells.Overall, the mean proportion of scar/normal left ventricular myocardiumwas 13% in rats receiving CD34+ cells compared with 36-45% for each ofthe other groups (p<0.01), FIG. 6 g.

Discussion

The experiments described above demonstrate that neo-angiogenesis of theinfarct bed by human bone marrow-derived endothelial cell precursorsprevents scar development, maintains viable myocardium, and improvesventricular function in a rodent model of myocardial ischemia. Followinginfarction, the viable myocardial tissue bordering the infarct zoneundergoes a significant degree of hypertrophy (5,34-35). Althoughneoangiogenesis within the infarcted tissue appears to be an integralcomponent of the remodeling process (36,37), under normal circumstancesthe capillary network cannot keep pace with tissue growth and is unableto support the greater demands of the hypertrophied, but viable,myocardium which subsequently undergoes apoptosis due to inadequateoxygenation and nutrient supply. The development of neoangiogenesiswithin the myocardial infarct scar appears to require activation oflatent collagenase and other proteinases following plasminogenactivation by urokinase-type plasminogen activator (u-PA) expressed oninfiltrating leukocytes (38). The importance of bone marrow-derivedendothelial precursors in this process has been demonstrated in u-PA −/−mice where transplantation of bone marrow from cogenic wild-type strainsrestored defective myocardial revascularization post-infarction (38).Since u-PA mRNA transcription and proteolytic activity in humanmononuclear cells and tumor cell lines is significantly increased by thecolony stimulating factors G-CSF, M-CSF, and GM-CSF (39-41), thisprovides a rationale for in vivo or ex vivo use of these cytokines tomobilize and differentiate large numbers of human adult bonemarrow-derived angioblasts for therapeutic revascularization of theinfarct zone.

Cell surface and RNA expression of the transcription factor GATA-2appears to selectively identify human adult bone marrow-derivedangioblasts capable of responding to signals from ischemic sties byproliferating and migrating to the infarct zone, and subsequentlyparticipating in the process of neo-angiogenesis. Of particularinterest, GATA-2 is a co-factor for endothelial cell transcription ofpreproendothelin-1 (ppET-1) (42), the precursor molecule of the potentvasoconstrictor and hypertrophic autocrine peptide ET-1. Since ppET-1transcription is also increased by angiotensin II (43), produced as aresult of activation of the renin-angiotensin neurohormonal axisfollowing myocardial infarction, the angioblasts infiltrating theinfarct bed may be secreting high levels of ET-1 due to the synergistincactions of angiotensin II surface receptor signalling and GATA-2transactivation. The observation that newly-formed vessels within theinfarct scar have thicker walls, lower vasodilator responses to strongervasoactive substances than vessels within normal myocardium (44) areconsistent with effects of increased autocrine ET-1 activity, andsupport the possibility that neo-angiogenic vasculature is derived frominfiltrating GATA-2 positive angioblasts.

Together, the results of the above-described experiments indicate thatinjection of G-CSF mobilized adult human CD34+ cells with phenotypic andfunctional properties of embryonic hemangioblasts can stimulateneo-angiogenesis in the infarct vascular bed, thus reducing collagendeposition and scar formation in myocardial infarction. Although thedegree of reduction in myocardial remodeling as a result ofneoangiogenesis was striking, further augmentation in myocardialfunction might be achieved by combining infusion of human angioblastswith ACE inhibition or AT₁-receptor blockade to reduce angiotensinII-dependent cardiac fibroblast proliferation, collagen secretion, andplasminogen activator-inhibitor (PAI) production (45, 46). The use ofcytokine-mobilized autologous human bone-marrow angioblasts forrevascularization of myocardial infarct tissue, in conjunction withcurrently used therapies (47-49), offers the potential to significantlyreduce morbidity and mortality associated with left ventricularremodeling post-myocardial infarction.

Second Series of Experiments

Methods

1. Purification of Cytokine-Mobilized Human CD34+Cells

Single-donor leukopheresis products were removed from humans treatedwith recombinant G-CSF 10 mg/kg (Amgen, CA) sc daily for four days.Mononuclear cells were separated by Ficoll-Hypaque, and highly-purifiedCD34+ cells (>98% positive) were removed using magnetic beads coatedwith anti-CD34 monoclonal antibody (mAb) (Miltenyi Biotech Ltd, CA).Purified CD34 cells were stained with fluorescein-conjugated mAbsagainst CD34, CD117, VEGFR-2, Tie-2, GATA-2, GATA-3, AC133, vWF, eNOS,CD54, CD62E, CXCR1, CXCR2, CXCR4, and analyzed by four-parameterfluorescence using FACScan (Becton Dickinson, CA).

2. Proliferative Studies of Human Endothelial Progenitors

Single-donor CD34-positive cells were cultured for 96 hours in RPMI witheither 20% normal rat serum, ischemic rat serum or 20 ng/ml VEGF, thenpulsed with [³H] thymidine (Amersham Life Science Inc, IL, USA) (1mlCi/well) and uptake was measured in an LK Betaplate liquidscintillation counter (Wallace, Inc., Gaithersburg, Md.). The proportionof CD117^(bright)GATA-2^(pos) cells after 96 hours of culture in eachcondition was also quantitated by flow cytometry.

3. Chemotaxis of Human Bone Marrow-Derived Endothelial Progenitors

Highly-purified CD34+ cells (>98% positive) were plated in 48-wellchemotaxis chambers fitted with membranes (8 mm pores) (Neuro Probe,MD). After incubation for 2 hours at 37°, chambers were inverted andcells were cultured for 3 hours in medium containing IL-8 at 0.2, 1.0and 5.0 mg/ml, SDF-1 alpha/beta 1.0 mg/ml, VEGF and SCF. The membraneswere fixed with methanol and stained with Leukostat (Fischer Scientific,Ill). Chemotaxis was calculated by counting migrating cells in 10high-power fields.

4. Animals, Surgical procedures, Injection of Human Cells, andQuantitation of Cellular Migration into Tissues

Rowett (rnu/rnu) athymic nude rats (Harlan Sprague Dawley, Indianapolis,Ind.) were used in studies approved by the “Columbia UniversityInstitute for Animal Care and Use Committee”. After anesthesia, a leftthoracotomy was performed, the pericardium was opened, and the leftanterior descending (LAD) coronary artery was ligated. Sham-operatedrats had a similar surgical procedure without having a suture placedaround the coronary artery. 48 hours after LAD ligation 2.0×10⁶DiI-labeled human CD34+ cells (>95%, 40%, <2% purity) removed from asingle donor after G-CSF mobilization were injected into the tail veinin the presence or absence of mAbs with known inhibitory activityagainst CXCR1, CXCR2, CXCR4, CD34, rat IL-8 (ImmunoLaboratories, Japan)and rat SDF-1® & D Systems, MN), or isotype control antibodies. Controlanimals received saline after LAD ligation. Each group consisted of 6-10rats. Quantitation of myocardial infiltration after injection of humancells was performed by assessment of DiI fluorescence in hearts fromrats sacrificed 2 days after injection (expressed as number ofDiI-positive cells per high power field, minimum 5 fields examined persample). Quantitation of rat bone marrow infiltration by human cells wasperformed in 12 rats at baseline, days 2, 7, and 14 by flow cytometricand RT-PCR analysis of the proportion of HLA class I-positive cellsrelative to the total rat bone marrow population.

5. Analyses of Myocardial Function

Echocardiographic studies were performed at baseline, 48 hours after LADligation, and at 2, 6 and 15 weeks after injection of cells or saline,using a high frequency liner array transducer (SONOS 5500, HewlettPackard, Andover, Mass.). 2D images were removed at mid-papillary andapical levels. End-diastolic (EDV) and end-systolic (ESV) leftventricular volumes were removed by bi-plane area-length method, and %left ventricular ejection fraction (LVEF) was calculated as[(EDV-ESV)/EDV]×100. Left ventricular area at the end of systole (LVAs)was measured by echocardiography at the level of the mitral valve. LVEFrecovery and reduction in LVAs were calculated as the mean improvementbetween the respective values for each at different time points afterLAD ligation relative to pre-infarct values.

6. Histology and Immunohistochemistry

Histologic studies were performed on explanted rat hearts at 2 and 15weeks after injection of human cells or saline. Following excision, leftventricles from each experimental animal were sliced at 10-15 transversesections from apex to base. Representative sections were put intoformalin for histological examination, stained freshly with anti-factorVIII mAb using immunoperoxidase technique to quantitate capillarydensity, or stained with Masson trichrome and mounted. The lengths ofthe infarcted surfaces, involving both epicardial and endocardialregions, were measured with a planimeter digital image analyzer andexpressed as a percentage of the total ventricular circumference. Finalinfarct size was calculated as the average of all slices from eachheart.

7. Measurement of Rat CXC Chemokine mRNA and Protein Expression

Poly(A)+ mRNA was extracted by standard methods from the hearts of 3normal and 12 LAD-ligated rats. RT-PCR was used to quantify myocardialexpression of rat IL-8 and Gro-alpha mRNA at baseline and at 6, 12, 24and 48 hours after LAD ligation after normalizing for total rat RNA asmeasured by GAPDH expression. After priming with oligo (dT) 15-mer andrandom hexamers, and reverse transcribed with Monoley murinelymphotrophic virus reverse transcriptase (Invitrogen, Carlsbad, Calif.,USA), cDNA was amplified in the polymerase chain reaction (PCR) usingTaq polymerase (Invitrogen, Carlsbad, Calif., USA), radiolabeleddideoxy-nucleotide ([α³²P]-ddATP: 3,000 Ci/mmol, Amersham, ArlingtonHeights, Ill.), and primers for rat IL-8, Gro-alpha and GAPDH (FisherGenosys, CA). Primer pairs (sense/antisense) for rat IL-8, Gro-alpha ANDGAPDH were, gaagatagattgcaccgatg (SEQ ID NO:1)/catagcctctcacatttc (SEQID NO:2), gcgcccgtccgccaatgagctgcgc (SEQ IDNO:3)/cttggggacacccttcagcatcttttgg (SEQ ID NO:4), andctctacccacggcaagttcaa (SEQ ID NO:5)/gggatgaccttgcccacagc (SEQ ID NO:6),respectively. The labeled samples were loaded into 2% agarose gels,separated by electrophoresis, and exposed for radiography for 6 h at−70°. Serum levels of rat IL-8/Gro-alpha were measured at baseline andat 6, 12, 24 and 48 hours after LAD ligation in four rats by acommercial ELISA using polyclonal antibodies against the rat IL-8/Grohomologue CINC (ImmunoLaboratories, Japan). The amount of protein ineach serum sample was calculated according to a standard curve ofoptical density (OD) values constructed for known levels of ratIL-8/Gro-alpha protein.

Experimental Procedures and Results

1. Selective Trafficking of Endothelial Precursors

Following immunoselection of G-CSF mobilized human CD34 cells to >98%purity, 60-80% co-expressed the stem cell factor receptor CD117. Byquadruple parameter analysis, FIG. 7 a, 10-15% of CD117^(bright) cellswere found to express a phenotype characteristic of embryonicangioblasts, with low level surface expression of VEGFR-2 and Tie-2, aswell as the transcription factors GATA-2 and GATA-3, and AC133, recentlyshown to identify endothelial precursors (79). These cells did notexpress markers of mature endothelial cells such as vWF, eNOS andE-selectin, but were positive for the CXC chemokine receptors 1, 2, and4. Intravenous injection of 2×10⁶ DiI-labeled human CD34+ cells (>98%,40%, and 2% purity) into LAD-ligated Rowett nude rats was accompanied at48 hours by dense infiltration of rat myocardium, FIG. 7 b. Thetrafficking of these cells was specifically directed to the infarct areasince few DiI-labeled cells were detected in unaffected areas of heartswith regional infarcts, not shown, and DiI-labeled cells did notinfiltrate myocardium from sham-operated rats, FIG. 7 b. By two weekspost-injection, rats receiving >98% pure human CD34+ cells demonstratedincreased infarct bed microvascularity and reduced matrix deposition andfibrosis, FIG. 7 c. The number of factor VIII-positive capillaries perhigh power field was over three-fold higher in the infarct bed of ratsreceiving 2×10⁶ cells containing >98% pure CD34+ purity than in theanalogous region in rats receiving 2×10⁶ cells containing either 2% or40% CD34+ purity, p<0.01, FIG. 7 c. Moreover, the majority of thesecapillaries were of human origin since they expressed HLA class Imolecules (not shown). Thus, although various populations of human bonemarrow-derived cells migrate to the infarct bed, vasculogenesis appearsto require selective trafficking of a critical number of endothelialprecursors.

2. Effects of Ischemia on CXC Chemokine Production by InfarctedMyocardium

Since human leukocyte chemotaxis and tissue infiltration is regulated byinteractions between specific chemokines and CXC cell surface receptors,we next investigated the effects of ischemia on CXC chemokine productionby infarcted rat myocardium. As shown in FIG. 8 a-c, infarctedmyocardium demonstrated a time-dependent increase in mRNA expression ofthe CXCR1/2-binding ELR-positive chemokines IL-8 and Gro-alpha, withmaximal expression at 6-12 hours after LAD ligation. In comparison tonon-infarcted myocardium, tissues after LAD ligation expressed 7.2-7.5fold higher mRNA levels of these ELR-positive pro-angiogenic chemokinesafter normalizing for total mRNA content (p<0.001). Moreover, serum IL-8levels increased by 8-10 fold within 6-12 hours after LAD ligation(p<0.001), and remained elevated at 48 hours, FIG. 8 d.Co-administration of blocking mAbs against either IL-8 and Gro-alpha, oragainst the surface receptors for these pro-angiogenic chemokines, CXCR1or CXCR2, reduced myocardial trafficking of human angioblasts by 40-60%relative to control antibodies (p<0.01), FIG. 8 e.

3. Chemotactic Responses of Human Bone Marrow-Derived CD34+Angioblaststo Chemokines.

In subsequent experiments we directly measured in vitro and in vivochemotactic responses of human bone marrow-derived CD34+ angioblasts toIL-8. As shown in FIG. 9 a, in vitro chemotaxis of human CD34+ cells wasinduced by IL-8 in a dose-dependent manner, with concentrations between0.2-5 μ/ml. The ELR− chemokine SDF-1, produced constitutively by bonemarrow stromal cells, induced a similar degree of chemotaxis of CD34+cells at concentrations similar to IL-8, FIG. 9 b. In contrast,chemotaxis was not induced by the growth factors VEGF or stem cellfactor (SCF). Moreover, intracardiac injection of IL-8 at 1 μg/ml intonon-infarcted hearts induced in vivo chemotaxis of CD34+ cells, FIG. 9c, whereas neither VEGF nor SCF, used as controls, had any chemotacticeffect in vivo, FIG. 9 d. Together, these results indicate thatincreased tissue expression of ELR-positive chemokines augmentsvasculogenesis in vivo by inducing chemotaxis of bone marrow-derivedendothelial precursor cells to sites of tissue ischemia.

4. Interruption of CXCR4/SDF-1 Interactions to Redirect Trafficking ofHuman CD34-Positive Cells from Bone Marrow to Myocardium.

In addition to augmenting trafficking of intravenously injected humanCD34+ angioblasts to damaged myocardium, ischemic serum from LAD-ligatedrats caused rapid expansion of the circulating CD34+CD117^(bright)angioblast population and concomitantly increased trafficking of thesecells to the bone marrow. As shown in FIG. 10 a, culture for 2 days witheither VEGF or ischemic serum increased proliferation ofCD34+CD117^(bright) angioblasts by 2.8 and 4.3 fold, respectively(p<0.01). Moreover, as shown in FIG. 10 b, bone marrow from ischemicrats after LAD ligation contained 5-8 fold higher levels of humanCD34+CD117^(bright) angioblasts compared with bone marrow from normalrats 2-14 days after intravenous injection of 2×10⁶ human CD34-positivecells (>95% purity), (p<0.001). Since SDF-1 is constitutively expressedby bone marrow stromal cells and preferentially promotes bone marrowmigration of circulating CD34+ cells which are actively cycling (80), weinvestigated whether the increased homing of human CD34+CD117^(bright)angioblasts to ischemic rat bone marrow was due to heightenedSDF-1/CXCR4 interactions. As shown in FIG. 10 c, co-administration ofmAbs against either human CXCR4 or rat SDF-1 significantly inhibitedmigration of intravenously administered CD34+ human angioblasts toischemic rat bone marrow by compared with anti-CD34 control antibody(both p<0.001). Moreover, co-administration of mAbs against either humanCXCR4 or rat SDF-1 increased trafficking of CD34+ human angioblasts toischemic rat myocardium by a mean of 24% and 17%, respectively (bothp<0.001), FIG. 10 d. By two weeks, the myocardial infarct bed of ratsreceiving human CD34+ cells in conjunction with anti-CXCR4 mAbdemonstrated >3-fold increase in microvascularity compared with thosereceiving CD34+ cells in conjunction with isotype control antibody.These results indicate that although intravenously injected CD34+angioblasts traffick to infarcted myocardium and induce vasculogenesisin response to augmented production of ELR+ chemokines, the efficiencyof this process is significantly reduced by concomitant angioblastmigration to the bone marrow in response to SDF-1. Interruption ofCXCR4/SDF-1 interactions redirects trafficking of the expanded, cyclingpopulation of human CD34-positive cells from bone marrow to myocardiumafter infarction, increasing infarct bed neoangiogenesis.

5. Improvement in Myocardial Function

Although left ventricular function was severely depressed after LADligation, injection of >98% pure CD34+ cells was associated withsignificant recovery in left ventricular size and function within twoweeks, and these effects persisted for the entire 15 week period offollow-up, FIGS. 11 a and b. In rats receiving >98% pure CD34+ cells,left ventricular end-systolic area decreased by a mean of 37±6% by 15weeks compared to immediately post-infarction, FIG. 11 a, and leftventricular ejection fraction (LVEF) recovered by a mean of 34±4% by 15weeks (p<0.001), FIG. 11 b (p<0.001). Improvement in these parametersdepended on the number of CD34+ cells injected, since intravenousinjection of 2×10⁶ G-CSF mobilized human cells containing 2% or 40%CD34+ purity did not significantly improve myocardial function despitesimilar degrees of trafficking to ischemic myocardium, FIGS. 11 a and b.However, co-administration of anti-CXCR4 mAb together with G-CSFmobilized human bone marrow-derived cells containing 40% CD34+ puritysignificantly improved LVEF recovery and reduced LVAs, to levels seenwith >98% CD34+ purity. By trichrome stain, significant differences inleft ventricular mass and collagen deposition were observed between thegroups, FIG. 11 c. In rats receiving 2×10⁶ human cells containing 2%CD34 purity, the left ventricular anterior wall was completely replacedby fibrous tissue and marked compensatory septal hypertrophy waspresent. Similar changes were seen in hearts of rats receiving 2×10⁶human cells containing 40% CD34 purity. In contrast, in hearts of ratsreceiving 2×10⁶ human cells containing 98% CD34 purity significantlygreater anterior wall mass was maintained, with normal septal size andminimal collagen deposition. Of particular interest, hearts of ratsreceiving 2×10⁶ human cells containing 40% purity together withanti-CXCR4 mAb demonstrated similar increase in anterior myocardial wallmass, decrease in septal hypertrophy, and reduction in collagendeposition.

Overall, the mean proportion of fibrous scar/normal left ventricularmyocardium was 13% and 21%, respectively, in rats receiving >98% pureCD34+ cells or 40% pure CD34+ cells together with anti-CXCR4 mAb,compared with 36-45% for rats receiving 2% and 40% pure CD34+ cells(p<0.01), FIG. 11 d. Thus, augmentation of infarct bed vasculogenesis byincreasing selective trafficking of a critical number of endothelialprecursors leads to further prevention of the remodeling process,salvage of viable myocardium, and improvement in cardiac function.

Discussion

This study demonstrates that ELR+ chemokines produced by ischemictissues regulate the development of compensatory vasculogenesis atischemic sites by producing a chemoattractant gradient for bonemarrow-derived endothelial cell precursors. Although both the ELR+ CXCchemokine IL-8 and the ELR− CXC chemokine SDF-1 demonstrate similareffects on chemotaxis of CD34+ endothelial precursors, as well as onmature endothelium (73), when expressed at different extravascular sitesthey impart opposing biological effects on directional egress ofendothelial progenitors, and consequently on tissue neovascularization.By understanding these interactions we were able to manipulate andaugment the chemotactic properties of a specific subset of human bonemarrow-derived CD34+ cells in order to increase myocardial trafficking,induce infarct bed vasculogenesis, reduce post-ischemic ventricularremodeling, and improve myocardial function.

Since migration of bone marrow-derived progenitors through basementmembrane is dependent on secretion of proteolytic enzymes such asmetalloproteinase-9 (MMP-9, Gelatinase B) (81), intracardiacmetalloproteinase activity may be a critical determinant of angioblastextravasation from the circulation and transendothelial migration intothe infarct zone. IL-8 induces rapid release (within 20 minutes) of thelatent form of MMP-9 from intracellular storage granules in neutrophils(82-83), and increases serum MMP-9 levels by up to 1,000-fold followingintravenous administration in vivo in non-human primates (84). SinceIL-8 significantly increases MMP-9 expression in bone marrow progenitors(81), and neutralizing antibodies against MCP-9 prevent mobilization ofthese cells (85), the results of our study suggest that angioblastinfiltration and subsequent infarct bed vasculogenesis may result fromIL-8-dependent increases in MMP-9 secretion.

Activation of latent MMP-9 and concomitant development ofneoangiogenesis within murine myocardial infarct scar tissue has beenshown to depend on urokinase-type plasminogen activator (u-PA)co-expressed by bone marrow progenitors infiltrating the infarct bed(81). Transcription and proteolytic activity of u-PA in human cells issignificantly increased by G-CSF and other colony stimulating factors(86-88). Since IL-8-induced chemotaxis and progenitor mobilizationrequire the presence of additional signals delivered through functionalG-CSF receptors (89), it is possible that increased u-PA activity isrequired for IL-8 mediated trafficking of angioblasts to sites ofischemia. This would explain the limited extent of infarct bedneoangiogenesis observed normally after myocardial infarction (62,63)despite high levels of IL-8 production, and provides a rationale for invivo or ex vivo administration of colony stimulating factors to mobilizeand differentiate human bone marrow-derived angioblasts for use intherapeutic revascularization of ischemic tissues.

Constitutive production of the CXC chemokine SDF-1 by bone marrowstromal cells appears to be essential for bone marrow homing andengraftment of haematopoietic progenitors (76-78). In addition,expression of SDF-1 in non-haematopoietic tissues plays a role in thedeveloping vascular system since SDF-1 −/− mice have defects in bothvascularization of the gastrointestinal tract (50) and ventricularseptum formation (90). Since bone marrow-derived endothelial precursorsexpress CXCR4 (80) and demonstrate chemotactic responses to SDF-1, asshown here, induced expression of SDF-1 at non-haematopoietic sitesduring embryogenesis or following tissue injury may be an importantelement in the process of tissue neovascularization (91). Our ability toredirect trafficking of human bone marrow-derived angioblasts to sitesof tissue ischemia by interruption of CXCR4/SDF-1 interactions arguesstrongly that SDF-1 is a biologically active chemotactic factor forhuman endothelial precursors, and that it may have pro-angiogenicactivity if expressed at non-haematopoietic sites. Future studies willaddress whether increased expression and localization of SDF-1 and otherchemokines at the sites of tissue ischemia might be synergistic withELR+ CXC chemokines in augmenting vasculogenesis. Together, the resultsof this study indicate that CXC chemokines, including IL-8, Gro-alpha,and SDF-1, play a central role in regulating human bone marrow-dependentvasculogenesis, and that manipulation of interactions between thesechemokines and their receptors on autologous human bone marrow-derivedangioblasts can enhance the potential efficacy of therapeuticvasculogenesis following tissue ischemia.

Third Series of Experiments

Experimental Procedures and Results

1. Myocardial Ischemia Induces Production of CC Chemokines and IncreasesHuman CD34+ Angioblast Expression of CC Chemokine Receptors

Since human mononuclear cell chemotaxis and tissue infiltration isregulated by interactions between cell surface receptors with specificchemokine ligands, the effects of ischemia on angioblast CC chemokinereceptor expression and on kinetics of CC chemokine production byinfarcted rat myocardium were investigated. As shown in FIG. 12, cultureof CD34+CD117^(bright) angioblasts with serum from LAD-ligated ratsincreased surface expression of CCR1 and CCR2, while surface expressionof CCR3 and CCR5 remained unchanged.

As shown in FIG. 13, infarcted myocardium demonstrated a time-dependentincrease in mRNA expression of several CCR-binding chemokines. Infarctedmyocardium was found to express over 8-fold higher levels of theCCR2-binding CC chemokine MCP-1, and 3-3.5-fold higher mRNA levels ofMCP-3 and RANTES, as well as the CCR3-binding chemokine eotaxin, afternormalizing for total mRNA content (all p<0.001). This pattern of geneexpression appeared to be relatively specific since every infarctedtissue studied demonstrated increased expression of these CC chemokinesand none demonstrated induced expression of the CCR5-binding CCchemokines MIP-1 alpha or MIP-1beta.

2. Trafficking of Human CD34+Angioblasts to Ischemic Myocardium isRegulated by Induced Expression of CC and CXC Chemokines

Next investigated was whether human angioblast trafficking to ischemicmyocardium was related to the induced expression of the CC chemokinesidentified above. Co-administration of blocking mAbs against MCP-1,MCP-3, and RANTES, or against eotaxin, reduced myocardial trafficking ofhuman angioblasts by 40-60% relative to control antibodies (p<0.01),FIG. 14. To prove that CC chemokines mediate angioblast chemotaxis toischemic myocardium, we measured in vivo angioblast chemotaxis inresponse to eotaxin. As shown in FIG. 15, intracardiac injection ofeotaxin into non-infarcted hearts induced 1.5-1.7 fold increase in CD34+angioblast trafficking whereas injection of the growth factors VEGF andstem cell factor had no effect on chemotaxis despite increasingangioblast proliferation (not shown).

Fourth Series of Experiments

Determination of Myocyte Size. Myocyte size was measured in normal rathearts and in the infarct zone, peri-infarct rim and distal areas ofinfarct tissue sections stained by trichrome. The transverse andlongitudinal diameters (mm) of 100-200 myocytes in each of 10-15high-powered fields were measured at 400× using Image-Pro Plus software.

Measurement of Myocyte Apoptosis by DNA End-Labeling of Paraffin TissueSections.

For in situ detection of apoptosis at the single cell level we used theTUNEL method of DNA end-labeling mediated by dexynucleotidyl transferase(TdT) (Boehringer Mannheim, Mannheim, Germany). Rat myocardial tissuesections were removed from LAD-ligated rats at two weeks after injectionof either saline or CD34+ human cells, and from healthy rats as negativecontrols. Briefly, tissues were deparaffinized, digested with ProteinaseK, and incubated with TdT and fluorescein-labeled dUTP in a humidatmosphere for 60 minutes at 370 C. After incubation for 30 minutes withan antibody specific for fluorescein conjugated alkaline phosphatase theTUNEL stain was visualized in which nuclei with DNA fragmentationstained blue.

1. Neoangiogenesis Protects Hypertrophied Myocardium Against Apoptosis.

The mechanism by which induction of neo-angiogenesis resulted inimproved cardiac function was investigated.

Results showed that two weeks after LAD ligation the myocytes in theperi-infarct rim of saline controls had distorted appearance, irregularshape, and similar diameter to myocytes from rats without infarction(0.020 mm +/−0.002 vs 0.019 mm +/−0.001). In contrast, the myocytes atthe peri-infarct rim of rats who received CD34+ cells had regular, ovalshape, and were significantly larger than myocytes from control rats(diameter 0.036 mm +/−0.004 vs 0.019 mm +/−0.001, p<0.01). Byconcomitant staining for the myocyte-specific marker desmin and DNAend-labeling, 6-fold lower numbers of apoptotic myocytes were detectedin infarcted left ventricles of rats injected with CD34+ cells comparedwith saline controls (apoptotic index 1.2+0.6 vs 7.1+0.7, p<0.01). Thesedifferences were particularly evident within the peri-infarct rim, wherethe small, irregularly-shaped myocytes in the saline-treated controlshad the highest index of apoptotic nuclei. In addition, whereasapoptotic myocytes extended throughout 75-80% of the left ventricularwall in saline controls, apoptotic myocytes were only detectable for upto 20-25% of the left ventricle distal to the infarct zone in ratsinjected with CD34+ cells. Together, these results indicate that theinfarct zone vasculogenesis and peri-infarct angiogenesis induced byinjection of CD34+ cells prevented an eccentrically-extendingpro-apoptotic process evident in saline controls, enabling survival ofhypertrophied myocytes within the peri-infarct zone and improvingmyocardial function.

2. Early Neoangiogenesis Prevents Late Myocardial Remodeling.

The last series of experiments showed the degree of peri-infarct rimmyocyte apoptosis at two weeks in control and experimental groups(saline vs CD34+ cells) compared with progressive myocardial remodelingover the ensuing four months. Despite similar initial reductions in LVEFand increases in LVAS, by two weeks the mean proportion of collagenousdeposition or scar tissue/normal left ventricular myocardium, as definedby Massonís trichrome stain, was 3% in rats receiving CD34+ cellscompared with 12% for those receiving saline. By 15 weekspost-infarction, the mean proportion of scar/normal left ventricularmyocardium was 13% in rats receiving CD34+ cells compared with 36-45%for each of the other groups studied (saline, CD34−, SVEC) (p<0.01).Rats receiving CD34+ cells demonstrated significantly increased mass ofviable myocardium within the anterior free wall which comprised myocytesexclusively of rat origin, expressing rat but not human MHC molecules,confirming intrinsic myocyte salvage rather than myocyte regenerationfrom human stem cell precursors. Whereas collagen deposition and scarformation extended almost through the entire left ventricular wallthickness in controls, with aneurysmal dilatation and typical EKGabnormalities, the infarct scar extended only to 20-50% of the leftventricular wall thickness in rats receiving CD34+ cells. Moreover,pathological collagen deposition in the non-infarct zone was markedlyreduced in rats receiving CD34+ cells.

Together, these results indicate that the reduction in peri-infarctmyocyte apoptosis observed at two weeks resulted in prolonged survivalof hypertrophied, but viable, myocytes and prevented myocardialreplacement with collagen and fibrous tissue by 15 weeks.

Discussion

The observation that proliferating capillaries at the peri-infarct rimand between myocytes were of rat origin shows that in addition tovasculogenesis human angioblasts or other co-administered bone-marrowderived elements may be a rich source of pro-angiogenic factors,enabling additional induction of angiogenesis from pre-existingvasculature.

References

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What is claimed:
 1. A method of increasing trafficking of endothelialprogenitor cells to an ischemic myocardium in a subject's heartcomprising administering to the subject's heart an amount ofStromal-Derived Factor-1 (SDF-1) effective to attract endothelialprogenitor cells to the ischemic myocardium so as to thereby increasetrafficking of endothelial progenitor cells to the ischemic myocardiumin the subject's heart.
 2. The method of claim 1, wherein the SDF-1 isadministered to heart muscle.
 3. The method of claim 1, wherein theSDF-1 is administered in a protein form.
 4. The method of claim 1,wherein the SDF-1 is administered by injection into the subject's heart.5. The method of claim 1, wherein the endothelial progenitor cells arebone marrow-derived endothelial progenitor cells.
 6. The method of claim1, wherein the SDF-1 is SDF-1 alpha.
 7. The method of claim 1, whereinthe SDF-1 is SDF-1 beta.
 8. The method of claim 1 further comprisingadministering endothelial progenitor cells to the subject's heart. 9.The method of claim 1, wherein the increased trafficking of endothelialprogenitor cells to the ischemic myocardium improves cardiac function inthe ischemic myocardium of the subject's heart.
 10. The method of claim1, wherein the subject has suffered or is suffering from one or more ofthe following: myocardial infarction, chronic heart failure, ischemicheart disease, coronary artery disease, diabetic heart disease,hemorrhagic stroke, or thrombotic stroke.
 11. The method of claim 1,further comprising administering to the subject one of more of thefollowing: an inhibitor of Plasminogen Activator Inhibitor, AngiotensinConverting Enzyme Inhibitor or a beta blocker.